1. Introduction
It is well established that many pathological conditions, including but not limited to infections, cancer, and autoimmune disorders, are characterized by aberrant expression of certain molecules. It is also known that the altered expression of these molecules leads to the induction of a humoral-immune response and the production of circulating antibodies against those molecules. Both the disease-associated molecules and the presence of serum antibodies are useful “markers” for the detection and monitoring of particular pathologies and for the generation of diagnostic and/or therapeutic agents.
2. Detection of Disease-associated Antibodies in Patient Samples
Although accurate assays for determining the presence of disease markers in a subject are commercially available, these assays rely on the detection of serum antibodies against particular markers and are both complex and inconvenient. There has long been a need for non-invasive, simple assays for antibodies specific for the presence of disease markers. Assays that rely on the detection of specific antibodies in urine, for example, are non-invasive, require less sample preparation and are generally less complex than serum-based assays. High-titer antibody responses have been detected in both serum and urine of HIV-, Helicobacter pylori-, Schistosoma mansoni-, Schistosoma haematobium-, and Schistosoma japonicum-infected individuals. (Wu et al. (2001) Hepatogastroenterology 48: 614–7; Zhu et al. (2000) Diagn. Microbiol. Infect. Dis. 38: 237–41; Yamamoto et al. (2000) Helicobacter 5: 160–4; Bauer et al. (1992) Lancet 340: 559). A strong correlation between serum-antibody and urine-antibody levels, combined with the beneficial features of urine-based assays compared to serum-dependent tests, makes urine-based diagnostics particularly attractive. Although the presence of antibody against infectious agents in urine has been reported (Wu et al. (2001) Hepatogastroenterology 48: 614–7; Zhu et al. (2000) Diagn. Microbiol. Infect. Dis. 38: 237–41; Yamamoto et al. (2000) Helicobacter 5: 160–4; Bauer et al. (1992) Lancet 340: 559), the utility of these methods for the detection of antibodies against autologous molecules such as tumor-associated antigens and self-antigens, in diagnostic methods for cancer or autoimmune disease, has never been demonstrated.
Both humoral- and cellular-immune responses against human tumor-associated antigens have been observed in patients with different types of cancer (Knuth et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3511–5; Sahin et al. (1995) Proc. Natl. Acad. Sci. USA 92: 11810–3). Based on the specific recognition by humoral and/or cellular effectors of the immune system, a large number of tumor-associated antigens—and thus, likely diagnostic reagents—have been identified (Knuth et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3511–5; Old (1981) Cancer Res. 41: 361–75; Angelopoulou et al. (1994) Int. J. Cancer 58: 480–7; Jager et al. (1998) J. Exp. Med. 187: 265–70; Tureci et al. (1997) Mol. Med. Today 3: 342–9). These antigens can be categorized into six groups according to their expression patterns, function or origin. Specifically, these groups include: a) Cancer-Testis (CT) antigens, which are expressed in normal germ cells and aberrantly expressed in different tumors (van der Bruggen et al. (1991) Science 254: 1643–7; Tureci et al. (1998) Proc. Natl. Sci. USA 95: 5211–6; Gure et al. (2000) Int. J. Cancer 85: 726–32; Gure et al. (1997) Int. J. Cancer 72: 965–71; Chen et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1914–8); b) differentiation antigens, which are expressed in tumor cells and the corresponding normal tissue of origin (Brichard et al. (1993) J. Exp. Med. 178: 489–95; Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3515–9; Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91: 6458–62; Jager et al. (2001) Cancer Res. 61: 2055–61); c) mutated normal genes (Disis et al. (1997) Adv. Cancer Res. 71: 343–71; Coulie et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7976–80; Wolfel et al. (1995) Science 269: 1281–4); d) overexpressed ‘self’ antigens (Disis et al. (1997) Adv. Cancer Res. 71: 343–71; Gnjatic et al. (1998) J. Immunol. 160: 328–33); e) viral antigens (Lennette et al. (1995) Eur. J. Cancer B31A: 1875–8; Tindle et al. (1996) Curr. Opin. Immunol. 8: 643–50); and f) splice variants of normal genes (Scanlan et al. (1998) Int. J. Cancer 76: 652–8; Jager et al. (1999) Cancer Res. 59: 6197–204).
The CT antigen NY-ESO-1, initially identified by serological expression cloning of a recombinant cDNA library obtained from a squamous cell carcinoma of the esophagus, elicits both humoral- and cellular immune responses in patients with NY-ESO-1-positive cancers (Chen et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1914–8; Jager et al. (1998) J. Exp. Med. 187: 265–70; Jager et al. (1999) Int. J. Cancer 84: 506–10; Stockert et al. (1998) J. Exp. Med. 187: 1349–54). NY-ESO-1 serum antibody is a reliable indicator of CD4- and CD8-positive T-cell responses against NY-ESO-1-derived peptide epitopes presented by different MHC class I and class II alleles (Chen et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1914–8; Jager et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4760–5; Jager et al. (2000) J. Exp. Med. 191: 625–30). High-titer NY-ESO-1 serum antibody was found in patients with advanced NY-ESO-1-positive malignancies. Changes in NY-ESO-1 antibody titers over extended periods of time correlate with the clinical development of NY-ESO-1-positive disease (Jager et al. (1998) J. Exp. Med. 187: 265–70).
As with NY-ESO-1, other tumor-associated antigens have been shown to elicit both humoral and cellular immune responses that are demonstrated by detection of both autologous antibodies and T cells in cancer patients. Examples of these tumor-associated antigens are SSX2 (Ayyoub et al. (2002) J. Immunol. 168: 1717–1722; Sahin et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 11810–11813; both incorporated herein by reference), MAGE-1 (Sahin et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 11810–11813; van der Bruggen et al. (1991) Science 254: 1643–1647; both incorporated herein by reference) and tyrosinase (Sahin et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 11810–11813; Brichard et al. (1993) J. Exp. Med. 173: 489–495; both incorporated herein by reference). In view of the finding that disease specific antibodies are detected in the urine of patients infected with various organisms (Wu et al. (2001) Hepatogastroenterology 48: 614–7; Zhu et al. (2000) Diagn. Microbiol. Infect. Dis. 38: 237–41; Yamamoto et al. (2000) Helicobacter 5: 160–4; Bauer et al. (1992) Lancet 340: 559), and the finding that—in addition to activating cellular immunity—tumor-associated antigens induce production of detectable levels of serum antibody, the inventors investigated whether aberrations in the expression of autologous proteins in non-infectious diseases and disorders, such as tumor-associated antigens, result in the excretion of antibodies urine. The results of these investigations provide the basis for the development of urine-based tests for the detection and monitoring of spontaneous and vaccine-induced immunity against defined disease-associated antigens in patients suffering from disorders characterized by the aberrant expression of the antigen.
The present invention demonstrates that specific antibodies against an aberrantly expressed molecule can be detected in urine samples taken from patients believed to be expressing that molecule, and that these molecules and their derivatives are useful diagnostic and therapeutic reagents. This discovery has important implications for the detection and monitoring of spontaneous and vaccine-induced immunity against defined disease-associated antigens.
3. Identification of Disease-associated Antigens: SEREX
The identification and preparation of disease-associated antigens is laborious, unpredictable and expensive. Two main methods have been used for the detection of such antigens. In the gene-based approach, host cells are transformed or transfected with a tumor-derived cDNA library and tested for the expression of the specific antigen. See, e.g., dePlaen et al. (1988) Proc. Natl. Sci. USA 85: 2275; incorporated herein by reference. In contrast, the biochemical approach is based on the elution of peptides from MHC-class I molecules of tumor cells, followed by isolation and purification of those peptides by reverse-phase high performance liquid chromatography (RP-HPLC) and testing for the ability of the peptides to bind MHC-class I molecules and activate cytotoxic T-lymphocytes (CTLs), including induction of CTL proliferation, release of tumor necrosis factor (TNF), and the lysis of target cells. The disadvantages of these approaches are highlighted by the relatively few new antigens identified by these methods. See, e.g., van der Bruggen et al. (1991) Science 254: 1643–1647; Brichard et al. (1993) J. Exp. Med. 178: 489–495; Coulie, et al. (1994) J. Exp. Med. 180: 35–42; Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3515–3519. Furthermore, these methods require established, permanent tumor-cell lines of the cancer type under consideration. Stable tumor-cell lines are very difficult to establish and maintain. See, e.g., Oettgen et al. (1990) Immunol. Allerg. Clin. North. Am. 10: 607–637. Also, numerous tumor types, including epithelial cell-tumors, are not responsive to CTLs in vitro, thereby precluding routine assay. These problems have led to the development of new methods for identifying disease-associated antigens.
The SEREX method (“Serological identification of antigens by Recombinant Expression Cloning”) has been used to both identify new disease-associated antigens and confirm expression of previously identified antigens. Sahin et al. (1995) Proc. Natl. Acad. Sci. USA 92: 11810–11913; Crew et al. (1995) EMBO J. 144: 2333–2340; U.S. Pat. Nos. 5,698,396; 6,251,603; and 6,252,052; each incorporated herein by reference. In brief, the SEREX method involves the expression of tumor-derived cDNA libraries in a prokaryotic host followed by screening with absorbed and diluted sera. The binding of serum antibodies to the expressed protein antigens identifies those proteins as antigens likely to elicit a high titer humoral-immune response. SEREX has led to the identification, isolation and cloning of a large number of immunogenic molecules that induce autologous humoral antibody responses. Several of these molecules have also been shown to induce autologous T-cell responses. NY-ESO-1 was identified by SEREX using serum from a patient with an esophageal tumor. See, e.g., U.S. Pat. Nos. 5,698,396; 6,251,603; 6,252,052; and U.S. patent application Ser. No. 09/062,422; each incorporated herein by reference. SEREX has also been used to monitor the status of a disease or disorder by assaying for antibodies specific to that disease or disorder. See, e.g., U.S. Pat. Nos. 5,698,396; 6,251,603; 6,252,052; and U.S. patent application Ser. No. 09/062,422 each incorporated herein by reference.
The search for disease- and tumor-associated antigens may be improved by screening disease- and tumor-derived cDNA expression libraries with autologous or allogeneic urine samples. The inventors have demonstrated that urine represents a source of highly specific, high-titer antibodies that are useful in diagnostic and therapeutic applications and in methods for identifying new disease-associated antigens.